Monoclonal antibodies specific to the human ldl receptor, their production and use

Description

(54) Monoclonal antibodies specific for human soluble LDL receptor, their preparation and use (57) Abstract The invention provides monoclonal antibodies that specifically recognize soluble human low-density lipoprotein receptors (LDLRs). These antibodies can be used, for example, in production processes for the identification and purification of human soluble LDLRs (hsLDLRs), and for the identification and treatment of diseases such as hepatitis C virus (HCV) infection. The invention also provides hybridomas that produce monoclonal antibodies of the invention, as well as uses and methods for preparing the antibodies of the invention. HU 226 194 B1 The description is 18 pages long (including 3 pages of illustrations). HU 226 194 B1 The invention provides monoclonal antibodies that specifically recognize soluble human low-density lipoprotein receptors (LDLRs). These antibodies can be used, for example, in production processes for the identification and purification of human soluble LDLRs (hsLDLRs), and for the identification and treatment of diseases such as hepatitis C virus (HCV) infection. The invention also provides hybridomas that produce monoclonal antibodies of the invention, as well as uses and methods for preparing the antibodies of the invention. Cholesterol is a component of the plasma membrane of all eukaryotes and is essential for the growth and viability of higher organisms. However, high serum cholesterol levels cause disease and death by promoting the formation of atherosclerotic plaques in arteries throughout the body. The major site of cholesterol synthesis in mammals is the liver. A measurable amount of cholesterol is also produced in the intestine. The rate of cholesterol formation in these organs is largely responsive to the amount of cholesterol absorbed from dietary sources. Extrahepatic and extraintestinal cells obtain cholesterol from the plasma rather than by de novo synthesis. Cholesterol and other lipids are transported in body fluids by lipoproteins, which are classified by their density. In the lipoprotein particle, a core of hydrophobic lipids is surrounded by a shell of polar lipids and apoproteins.These lipoproteins have two functions: they solubilize highly hydrophobic lipids and they carry signals that regulate the movement of these lipids into and out of specific target cells and tissues. Cholesterol is transported in body fluids by low-density lipoproteins (LDL), which bind to specific receptors on the plasma membrane of non-hepatic cells. The receptor-LDL complex is then internalized into the cell by a transport mechanism known as receptor-mediated endocytosis (Goldstein, 1979). The low-density lipoprotein (LDL) receptor is a prototype of a family of structurally related cell surface receptors that mediate the endocytosis of multiple ligands in mammalian cells. The LDL receptor consists of 822 amino acids and has a molecular weight of 164,000. It is composed of several domains, some of which show sequence homology to other proteins. Its N-terminal ligand-binding domain consists of 292 amino acids, which are arranged in 7 cysteine-rich repeats. Each repeat contains six cysteines, which are disulfide bonded in the following pattern: first to third, second to fifth, and fourth to sixth (Bieri et al., 1995). This domain is followed by four additional domains: the first consists of 400 amino acids and is homologous to the EGF receptor, the second consists of 58 amino acids rich in O-linked sugars, the third is a single transmembrane domain of 22 amino acids, and the fourth is a cytoplasmic domain of 50 amino acids (Sudhof et al., 1985; Brown et al., 1986). The physiological significance of the LDL receptor was established by Brown and Goldstein in their study of hereditary hypercholesterolemia (FH). They found that the disease is the result of a molecular genetic defect caused by the absence or deficiency of a functional LDL receptor (Brown et al., 1976). Several FH mutations have been characterized (Goldstein et al., 1975). A soluble form of sLDLR, which exhibits antiviral activity, has been identified and isolated from the culture supernatant of interferon-induced cells (Fischer et al., 1993) and from body fluids (Fischer et al., 1994). Several interferon-induced proteins have been identified that play a role in the antiviral state induced by IFNs. One such protein, which exhibits antiviral activity, has been produced and accumulated in the culture supernatant of WISH cells derived from human amnion (fetal envelope). This protein has been purified to homogeneity and identified as sLDLR (see European Publication No. EP 0 533 667 and Fischer et al., 1993). It has been found that sLDLR is secreted into the medium by mammalian cells that have entered an antiviral state in response to interferon. Unlike interferon, sLDLR does not induce an antiviral state in cells, but is itself antiviral.It has been found that sLDLR appears to be present at all stages of the viral replication maturation and assembly process, suggesting that it may play a role in the complex process that leads to inhibition of viral assembly or assembly (data not published). Hepatitis C virus endocytosis has recently been shown to be mediated by LDL receptors in cultured cells (Agnello et al., 1999). These and other findings suggest that the LDL receptor family may serve as virus receptors. Therefore, antibodies raised against sLDLR receptor may block the entry and assembly of viral particles through binding to the cellular LDL receptor. The only known monoclonal antibody specific for LDLR available is C7, a bovine LDLR-specific antibody (Beisiegel et al., 1981, commercially available from Amersham), which was prepared by immunizing mice with homogeneously purified bovine adrenal cortex LDLR. Bovine adrenal cortex membranes were solubilized and the receptor was partially purified by elution from a DEAE-cellulose column (Beisiegel et al., 1981). The bovine LDLR-specific antibody cross-reacted only weakly with human LDLR. In fact, it was found that C7-Mab raised against bovine LDLR had significant disadvantages when used to detect and quantify recombinant human LDLR: a) showed very low affinity for human LDLR, b) gave significant cross-reaction with contaminants in cell culture. HU 226 194 B1 Antibodies specific for human LDLR are not yet available. This seems surprising, since it is very common to raise antibodies against new proteins for purification, identification, or assay development. It is possible that such antibodies have not been produced so far because the production of monoclonal antibodies requires the availability of sufficiently large amounts of highly purified antigen to allow efficient immunization of mice. Highly purified antigen appears as a single, major peak in RP-HPLC. Furthermore, the identification and quantification of the antigen during the purification process is not easy to develop. In the present invention, the described antiviral assay was used to identify LDLR during the purification process. There is a need for the production of Mabs specific for human soluble LDLR, which would allow the development of an efficient immunoassay (ELISA) and the identification of the protein by Western blot. These antibodies are needed during the development of recombinant protein production and purification processes, for the tracking and measurement of recombinant human soluble LDLR, and for the detection of the native protein. The solution according to the invention enables the production of hybridoma cell lines that produce monoclonal antibodies capable of specifically recognizing and binding human LDL receptor and its fragments. More specifically, the solution according to the invention enables the production of hybridoma cell lines that produce monoclonal antibodies capable of specifically recognizing and binding human soluble LDL receptor. Thus, the invention provides a monoclonal antibody, chimeric antibody, humanized antibody, anti-anti-ld antibody or fragment thereof, which is capable of specifically recognizing and binding human soluble LDL receptor and fragments thereof. The invention provides monoclonal antibodies that are capable of recognizing and binding human soluble LDLR and that meet the following requirements: 1. Mabs that can be used as a pair in ELISA, e.g. sandwich ELISA ("Enzyme Linked immuno Sorbent Assay") for the detection of human, soluble LDLR. 2. Mabs that can be used to identify LDLR in Western blot analysis. 3. Mabs that can be used to neutralize the antiviral biological activity of human soluble LDLR. 4. Mabs that can be used to inhibit viral infection, such as HCV. The invention also provides a method for detecting and / or quantifying human LDLR, in which specific monoclonal antibodies according to the invention are used in a known manner for this purpose. The invention also provides a cloned hybridoma comprising a splenocyte and a homogeneous or heterogeneous lymphoid cell derived from a mammal immunized with recombinant human LDLR. The monoclonal antibody of the invention is prepared by conventional methods, for example by growing a cloned hybridoma containing a splenocyte and a homogeneous or heterogeneous lymphoid cell from a mammal immunized with recombinant human LDLR in a liquid medium or in the peritoneal cavity of a mammal, which allows the hybridoma to produce and accumulate the monoclonal antibody. In another aspect, the invention provides a method for purifying human LDLR, comprising contacting a material containing crude LDLR with a monoclonal antibody of the invention. A column adsorbed with a monoclonal antibody specific for LDLR may be used as an affinity purification step in a process for purifying the recombinant protein. In a method for detecting and measuring recombinant human LDLR, the monoclonal antibody of the invention is used as the antibody in an ELISA assay, as described in Example 5. Any LDLR can be used as an LDLR or fragment thereof for immunization of animals, provided that it is an LDLR from a warm-blooded animal. A mutein of LDLR can also be used. A representative example of such a mammalian soluble human LDLR is the soluble LDLR +291 form, which comprises an amino acid sequence starting at the Asp amino acid at position +4 of the human LDLR sequence and ending at the Glu amino acid at position +291, and any other form can be used, such as the +292 form, etc. Figure 1 shows a flow chart for the production of monoclonal antibodies against r-hsLDLR. Figure 2 shows Western blot analysis of the following materials: r-hsLDLR +291 form in lane 1, urinary hsLDLR in lane 2 and recombinant human p55 TNF receptor (r-hTBP-1) as a negative control in lane 3, using the monoclonal antibodies indicated below each test strip. The arrows on the left side of the figure indicate the positions of the molecular weight markers, and the arrows on the right side of the figure indicate the positions of the hsLDLR form, which are indicated above and below the arrows. Figure 3 shows the effect of Mabs 12.6, 28 and 29.8 on the production of HCV(+) and (-) strands in FT167 culture. Cells were treated with anti-LDLR-Mab for 30 min before infection (8 or 2 pg / ml). Cells were then infected overnight with 25 µl of HCV(+) serum (N°42; 1b). The day after infection, three washes were performed and fresh medium was added and changed every 48 h. HU 226 194 B1 Five days after infection, hepatocytes were isolated, RNA was purified, and 1 pg of RNA was analyzed by rTh-RTPCR (Perkin-Elmer). The assay procedures were performed in duplicate. +SP: positive strand RNA assay; -SP: negative strand RNA assay; X: blank. We have produced monoclonal antibodies specific for human soluble LDLR (hsLDLR). Using these monoclonal antibodies, we have developed ELISA and Western blot methods for the identification of hsLDLR, and an assay for neutralizing the antiviral activity of hsLDLR. Mabs were produced in mice immunized with recombinant +291 form of hsLDLR, which contains the N-terminal ligand binding domain of human soluble LDLR from Asp+4 to Glu+291. The recombinant +291 form of hsLDLR was produced in CHO cells and purified to homogeneity. Specific antibodies were produced in significant titers in the immunized mice. After screening of hybridomas, five clones (no. 12, 28, 29, 30 and 50) were identified that produced the highest levels of antibodies. These clones were selected for further subcloning. After subcloning, 29 highly productive subclones were isolated, and the parental clones and subclones were frozen in vials. A pair of monoclonal antibodies were selected for ELISA for the measurement of r-hsLDLR. Mab28 was selected as the capping antibody and biotin-labeled Mab29.8 was selected as the secondary antibody. Mab12.6 and 29.8 were found to be suitable for the identification of native and recombinant hsLDLR by Western blot analysis, and Mab12.6 and 50.30 were found to be suitable for the identification of recombinant hsLDLR by Western blot analysis. During the development of the solution according to the invention, it was found that Mabs 12.6, 28 and 29.8 also inhibit the replication of the hepatitis C virus (HCV) viral genome in primary cultures of human hepatocytes. Therefore, these antibodies can be used for the treatment of hepatitis C infection (Figure 3). The subclass isotype of the Mabs produced by the clones was determined. Clones 12.6, 28, 29.8 and 30 were identified as IgGr, while clone 50.30 was found to be IgM. Mabs raised against the +291 form of hsLDLR are also able to recognize other forms of hsLDLR, i.e. the +292 form and the +331 form of hsLDLR produced in recombinant CHO cells in ELISA and Western blot analysis. The +292 form contains the N-terminal part of the receptor from amino acid Asp+4 to Cys+292, and the +331 form contains the N-terminal part of the receptor from amino acid Asp+4 to Cys+331. The antigen used to produce monoclonal antibodies and immunize mice was the +291 form of r-hsLDLR, which was produced in CHO cells. The r-hsLDLR production was carried out in bioreactors using a stationary phase Fibracel matrix system. The r-hsLDLR was purified to homogeneity and used to immunize mice. Immunospleen cells from the best responding mouse were used for fusion and generation of hybridomas. With respect to the antibodies mentioned herein, the term "monoclonal antibody" is intended to mean monoclonal antibodies, chimeric antibodies, fully humanized antibodies, antibodies specific for anti-idiotypic antibodies (anti-anti-ld antibody), which may be labeled in soluble or bound form, as well as fragments thereof produced by any known technique, such as enzymatic cleavage, peptide synthesis or recombinant techniques. A monoclonal antibody (abbreviated as Mab) comprises a population of substantially homologous antibodies specific for antigens, which populations contain substantially similar epitope binding sites. Mabs can be prepared by methods known to those skilled in the art. See, for example, Kohler and Milstein, Nature 256, 495-497 (1975); U.S. Patent No. 4,376,110; Ausubel et al., eds. Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory (1988); and Colligan et al., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY (1992-1996), the entire contents of which references are hereby incorporated by reference. Such antibodies may be of any antibody class, for example, IgG, IgM, IgE, IgA, GILD, and any subclass thereof. The hybridoma-producing mAb of the invention may be cultured in vitro, in situ, or in vivo.The production of high titers of Mabs in vivo or in situ currently allows for an advantageous production process. Chimeric antibodies are molecules that have different parts derived from different animal species, for example, those that have a variable region derived from a murine Mab and a constant region derived from a human immunoglobulin. Chimeric antibodies are primarily used to reduce immunogenicity and increase production yield, for example, if the yield of murine Mabs from hybridomas is higher but highly immunogenic in humans, then human / murine chimeric Mabs are used. Methods for producing chimeric antibodies are well known to those skilled in the art [Cabilly et al., Proc. Natl. Acad. Sci. USA 81, 3273-3277 (1984); Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6851-6855 (1984); Boulianne et al., Nature 312, 643-646 (1984); EP 125023 (published November 14, 1984), Neuberger et al., Nature 314, 268-270 (1985); Taniguchi et al., EP 171496 (published February 19, 1985).); Morrison et al., EP 173494 (published March 5, 1986); Neuberger et al., PCT International Publication No. WO 86 / 01533; Kudo et al., European Publication No. EP 184187 (published June 11, 1986); Sahagan et al., J. Immunol. 137, 1066-1074 (1986); Robinson et al., International Publication No. WO 87 / 02671 (published May 7, 1987); Liu. HU 226 194 Β1 et al., Proc. Natl. Acad. Sci. USA 84, 3439-3443 (1987); Sun et al., Proc. Natl. Acad. Sci. USA 84, 214-218 (1987); Better et al., Science 240, 1041-1043 (1988); Riechmann et al., Nature 332, 323-327; and Harlow and Lane, “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory (1988)]. These references are hereby incorporated by reference in their entirety. “Fully humanized antibodies” are molecules in which both the variable and constant regions are derived from human immunoglobulin. Fully humanized antibodies have the potential to be used for therapeutic purposes where repeated treatments are required for chronic and relapsing disease, such as autoimmune diseases. One method for producing fully humanized antibodies consists of “humanizing” the humoral immune system of a mouse, i.e., creating a strain of mouse capable of producing human Ig (xenomouse), by introducing human immunoglobulin (Ig) loci into a mouse in which the endogenous Ig genes have been inactivated. Ig loci are extremely complex in terms of their physical structure and gene arrangement, and expression processes are required to ultimately elicit a broad-spectrum immune response.Antibody diversity is primarily generated by combinatorial arrangements between different V, D, and J genes in the Ig loci. These loci also contain regulatory elements, interspersed throughout, that regulate antibody expression, allelic exclusion, class switching, and affinity maturation. The introduction of disordered human Ig transgenes into mice has shown that the recombination mechanism operating in mice is compatible with human genes. Furthermore, hybridomas secreting antigen-specific hu mAbs of different isotypes have been generated by xenomice immunized with antigen. Fully humanized antibodies and methods for their production are well known in the art [Mendez et al., Nature Genetics 15, 146-156 (1997); Buggemann et al., Eur. J. Immunol. 21, 1323-1326 (1991); Tomizuka et al., Proc. Natl. Acad. Sci. USA 97, 722-727 (2000); International Publication No. WO 98 / 24893]. An anti-idiotypic (anti-Id) antibody is an antibody that recognizes unique determinants normally associated with the antigen-binding site of the antibody. An anti-Id antibody can be prepared by immunizing an animal of the same species and genetic type (e.g., mouse strain) as the source of the Mab with the MAb against which it is intended to produce anti-Id. The immunized animal recognizes and responds to the idiotypic determinants of the immunizing antibody by producing antibodies to the idiotypic determinants (anti-Id antibodies). See, for example, U.S. Patent No. 4,699,880, which is incorporated herein by reference in its entirety. The anti-Id antibody can also be used as an "immunogen" to induce an immune response in another animal, to produce a so-called anti-anti-Id antibody. The anti-anti-Id is epitopically identical to the original MAb against which the anti-Id was raised. Thus, by using antibodies specific for the idiotypic determinants of the MAb, other clones can be identified that express antibodies with the same specificity. Accordingly, Mabs of the invention raised against LDLR, its isoforms, analogs, fragments or derivatives can be used in animals suitable for inducing anti-ld antibodies, such as BALB / c mice. Spleen cells from such immunized mice are used to produce anti-ld hybridomas that secrete anti-ld Mabs. Furthermore, anti-ld Mabs can be coupled to a carrier, such as keyhole limpet hemocyanin (KLH), which can be used to immunize additional BALB / c mice. Sera from these animals contain anti-anti-ld antibodies that have the original Mab binding properties specific for an epitope of the LDLR protein, or its analogs, fragments and derivatives. Anti-ld Mabs therefore have their own idiotypic epitopes, or "idiotopes", which are structurally similar to the epitopes to be analyzed. The term "monoclonal antibody" refers to intact molecules as well as fragments thereof, such as Fab and F(ab')2, which are capable of binding antigen. Fab and F(ab')2 fragments lack the Fc fragment of the intact antibody, are cleared from the circulation more rapidly, and bind nonspecifically to tissues to a lesser extent than intact antibody [Wahl et al., J. Nucl. Med. 24, 316-325 (1983)]. It is understood that Fab and F(ab')2 and other antibody fragments can be used in the methods of the invention to detect and quantify a protein that interacts with LDLR protein, in accordance with the methods described for intact antibody molecules. Such fragments are typically prepared by protease cleavage using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). A monoclonal antibody is said to be "capable of binding a molecule" if it is capable of reacting specifically with the molecule, thereby causing the molecule to bind to the antibody. The term "epitope" as used herein refers to any portion of a molecule that an antibody can bind and that the antibody can recognize. Epitopes or "antigenic determinants" are usually chemically active, surface-bound groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural properties and specific charge properties. An “antigen” is a molecule or molecular fragment that is capable of being specifically bound by an antibody and which antigen is further capable of inducing the production in an animal of an antibody that is capable of binding an epitope of that antigen. An antigen may have one or more epitopes. The specific reaction referred to above indicates that5 HU 226 194 Β1 below, the antigen reacts highly selectively with the antibody that corresponds to it, and will not react with many other antibodies that have been produced against other antigens. Antibodies, including antibody fragments, can be used to quantitatively or qualitatively detect LDLR proteins in a sample, or to detect the presence of cells that express LDLR proteins of the invention. This can be accomplished by immunofluorescence techniques, which employ fluorescently labeled antibodies (see below) coupled to light microscopy, flow cytometry, or fluorimetric detection. The antibodies (or fragments thereof) of the invention can be used in histological studies, for example, in immunofluorescence or immunoelectron microscopy, to detect LDLR proteins of the invention in situ. In situ detection can be performed by taking a histological sample from a patient and adding a labeled antibody of the invention to this sample. The antibody (or fragment) is preferably added to the biological sample, or the labeled antibody (or fragment) is layered onto the biological sample. Using such a method, not only the presence of LDLR proteins can be determined, but also its distribution in the tissue under examination. Using the method of the invention, it will be obvious to the skilled person that any of the wide variety of histological methods (e.g., staining methods) can be modified for in situ detection. Such methods for assaying LDLR proteins according to the invention typically involve incubating a biological sample, such as a biological fluid, a tissue extract, freshly isolated cells, such as lymphocytes or leukocytes, or cells incubated in tissue culture, in the presence of a labeled antibody capable of identifying LDLR proteins, and detecting the antibody by any of a number of techniques well known to those skilled in the art. The biological sample may be coupled to a solid phase support or other carrier, such as nitrocellulose or other solid phase or other carrier capable of immobilizing cells, cell particles, or soluble proteins. The solid phase or other carrier is then washed with suitable buffers and then treated with detectably labeled antibody according to the invention, as described above. The solid phase or other carrier is then washed a second time with the buffer to remove unbound antibody. The amount of label bound to the solid phase or other carrier can then be detected by conventional means. The terms "solid phase support", "other support" or "carrier" are intended to refer to any support capable of binding antigen or antibodies. Well-known supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon amylases, natural and modified celluloses, polyacrylamides, gabbros and magnetite. The nature of the support may be soluble or insoluble to some extent for the purposes of the invention. The support may have virtually any possible structural configuration so long as the conjugated molecule is capable of binding antigen or antibody. Thus, the configuration of the solid phase or other support may be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. In other methods, the surface may be flat, such as a flat sheet, test strip, etc. Preferred solid phase or other supports include polystyrene beads.Many other suitable carriers for binding antibodies or antigens are known to those skilled in the art, or can be found through routine experimentation. The binding activity of a given batch of antibodies according to the invention can be determined according to well-known methods. One skilled in the art will be able to determine the effective and optimal conditions of the assay for each determination by performing routine experiments. In addition to washing, mixing, shaking, filtering, and the like, other steps may be introduced into the test procedure if customary and necessary in the given situation. One option for labeling an antibody in accordance with the invention is to conjugate the antibody to an enzyme and use it in an enzyme immunoassay (EIA). This enzyme, in turn, when contacted with a suitable substrate, will react with the substrate to form a group detectable by, for example, spectrophotometric, fluorimetric or visually. Enzymes that can be used for detectably labeling an antibody include, for example, malate dehydrogenase, Staphylococcus nuclease, delta-5 steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Detection can be performed by colorimetric methods, in which the chromogenic substrate of the enzyme is used.Detection can also be performed visually by comparing the extent of the enzyme reaction of a substrate with similarly prepared standards. Detection can be accomplished using any of a variety of other immunoassay methods. For example, R-PTPase can be detected by radioactive labeling of antibodies or antibody fragments in a radioimmunoassay (RIA). A thorough description of RIA can be found in Laboratory Techniques and Biochemistry in Molecular Biology, Work, T. S. et al., North Holland Publishing Company, NY (1978), especially the chapter entitled "An Introduction to Radioimmuno Assay and Related Techniques" by Chard, T. Irt, which is considered part of the teaching. A radioactive isotope can be detected, for example, in a gamma counter or scintillation counter or by autoradiography. The antibody of the invention can also be labeled with a fluorescent compound. If the fluorescently labeled antibody is exposed to light of an appropriate wavelength, it can be detected based on fluorescence. HU 226 194 Β1 week. The most commonly used fluorescent labeling compounds are: fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde and fluorescamine. The antibody can also be detectably labeled with fluorescent emitting metals, such as 152Eu or other metals of the lanthanide series. These metals can be coupled to the antibody using metal complexing groups, such as diethylenetriaminepentaacetic acid (ETPA). The antibody can also be detectably labeled by coupling it to a chemiluminescent group. The chemiluminescent labeled antibody can then be detected by the presence of luminescence, which is generated during the chemical reaction. Particularly useful chemiluminescent labeling groups include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester. Similarly, a bioluminescent group can be used to label the antibody of the invention. Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein enhances the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein can be detected by the presence of luminescence. Examples of important bioluminescent compounds for labeling purposes include luciferin, luciferase, and aequorin. The antibody molecule of the invention can be adapted for use in an immunometric assay, also known as a “two-site” or “sandwich” assay. In a typical immunometric assay, unlabeled antibody (or antibody fragment) is coupled to a solid support and detectably labeled soluble antibody is added, allowing the detection and / or measurement of the ternary complex formed between the solid phase, the antigen, and the labeled antibody. Typically and preferably, immunometric assays include "forward" assays in which the antibody bound to the solid phase is first contacted with the sample to be tested to extract the antigen from the sample, forming a binary complex of antibody and antigen on the solid phase. After a suitably selected incubation period, the solid support is washed to remove residual liquid sample and unreacted antigen (if any), and then contacted with a solution containing an unknown amount of labeled antibody (which acts as a "reporter molecule"). During a second incubation period, the labeled antibody may complex with the antigen bound to the solid support through the unlabeled antibody, and the solid support is then washed again to remove unreacted labeled antibody. Another “sandwich” type assay, which can also be used for the antigens of the invention, is the so-called “simultaneous and “reverse” assay. The “simultaneous” assay involves a single incubation step in which both the antibody coupled to the solid or other support and the labeled antibody are added to the sample to be tested simultaneously. After the incubation is complete, the solid or other support is washed to remove any remaining liquid sample and any uncomplexed labeled antibody. The presence of the labeled antibody associated with the solid or other support is then determined, similar to the conventional “forward” sandwich assay. In the “reverse” assay, a solution containing labeled antibody is added stepwise to the liquid sample, followed by the addition of unlabeled antibody bound to a solid or other carrier after a suitably selected incubation time. After a second incubation, the solid phase is washed in the usual manner to remove any remaining sample to be tested and any unreacted labeled antibody solution. The presence of the labeled antibody associated with the solid or other carrier is then determined, similar to the “simultaneous” and “forward” sandwich assay procedures. The solution according to the invention is now illustrated by the following examples, without thereby limiting the scope of the claimed protection. Example 1 Production of CHO r-hsLDLR Stable recombinant CHO cells expressing human soluble LDLR were generated by cotransfecting CHODUKS cells lacking the dihydrofolate reductase (DHFR) gene with two expression vectors (Urlaub, G. et al., 1980): psLDR01, which contains the N-terminal ligand-binding domain of LDLR from Asp (+4) to Asp (+291), and pDHFR, which contains the murine DHFR gene, both driven by the SV40 early region promoter and transcription termination elements. Transfection was performed with cationic liposomes using LipofectAmine (Gibco BRL) according to the manufacturer's protocol. 72 hours after transfection, the cells were transferred to selective medium containing 10% dialyzed FCS and free of deoxy- and ribonucleosides. Cells expressing DHFR activity were able to form colonies, which were isolated by picking the cells from trypsin-soaked paper discs.The cells were grown and screened for r-hsLDLR activity. The transfected cells were then subjected to gene amplification with MTX, then subcloned and stably producing clones were selected. r-hsLDLR (+291-form) was produced in a 5-liter CelliGen bioreactor in serum-free medium (Gibco CHO-A-SFM, cat. no. 95-0091 DJ) from a stable CHO-producing clone (designated #33-10-29-21). The crude fermentation broth was purified by filtration through a 0.8-2 μ cartridge filter (Gelman cat. no. CSS92DSCCK) and concentrated to 100% on a 5 kDa membrane. The +291-form of r-hsLDLR was used for the first immunizations, which was purified using a small-scale purification procedure. In this procedure, a DEAE-Sepharose cation exchange column was used, which was designed for hydrophobic interaction. HU 226 194 Β1 based step was followed on a Butyl-TSK column, followed by HTP column and size exclusion chromatography (SEC) on a Sephacryl-100 column. Fraction 27 of the SEC was selected because it contained up to 780 units of specific antiviral activity, detected by the antiviral assay described in Example 9 below. In this fraction, the protein was detected as r-hsLDLR by N-terminal analysis. A second batch of CHO +291 r-hsLDLR was purified and used for injection into mice. It was purified using a refined procedure to achieve improved yields, which consisted of the following steps: a) purification (by filtration) and concentration of the crude fermentation broth to 100%; b) HQ-POROS anion exchange column; and c) two hydrophobic interaction (HIC)-based steps: binding on a Butyl-TSK column and flow-through Phenyl-5PW column. The unbound fraction from the last HIC step was dialyzed and purified on an HS-POROS cation exchange column. The final step was performed on a hydroxyapatite (HTP) column. The r-hsLDLR thus produced was purified to approximately 90%, eluting as a single major peak in RP-HPLC. Example 2 Immunization of mice pg, purified r-hsLDLR, fraction 27 from the SEC column described above in Example 1, was homogenized at a concentration of 100 pg / ml with complete Freund's adjuvant (CFA, 50% v / v) and injected into the footpads of 5 7-week-old Balb / C female mice. Four weeks after the first immunization, mice were boosted intramuscularly with 10 pg of the same purified r-hsLDLR in 50% v / v CFA. Two weeks after the second injection, antisera from mice were tested for r-hsLDLR antibodies using the direct ELISA described in Example 3 below. Two mice with the most significant specific immunoreactivity, designated M-1 and M-2, were boosted with r-hsLDLR 10 weeks after the second injection, administered 10 pg of purified r-hsLDLR obtained in the refined purification procedure described above in Example 1. Mice were bled 14 weeks later and tested for antibodies specific for r-hsLDLR. Two additional booster doses of 50 pg r-hsLDLR in PBS were then given: the first intraperitoneally and the second two days later both intraperitoneally and intravenously. Two weeks after the second vaccination, the mice were bled and the antisera were tested for anti-r-hsLDLR activity using a direct ELISA as described in Example 3 below. Serial dilutions of each antisera were prepared from 1:100 to 1:32,000 and, in duplicate, 96-well plates were coated with 10 U / well of purified r-hsLDLR obtained by the refined purification procedure described in Example 1 above. The buffer used in the assay and DMEM+10% HS-containing PBS+1% BSA or gelatin+0.05% Tween 20+0.05% thimerosal were used as a blinded test in the first well of each row. Normal mouse serum (NMS) was added to the last two rows at the same dilution range as a negative control. The absorbance of the enzyme reaction was measured in an ELISA reader 4 92 and 405 sq m. The results of the test showed that the serum of the mouse designated M-1 had the highest immunoreactivity specific for r-hsLDLR, and therefore this mouse was sacrificed and its spleen cells were harvested for fusion with myeloma cells (Eshar, Z., 1985). Example 3 Direct ELISA for testing antisera and screening hybridoma clones Direct assay for screening positive antisera ELISA was performed as follows: 96-well plates were coated with 100 µl of r-hsLDLR (purified by the refined purification procedure described in Example 1) at a concentration of 100 units / ml (10 U / well) in PBS+1% gelatin (Sigma, cat. no. G-7765)+0.9 mM Ca2+ and 0.5 mM Mg2+, pH=5.6, hereafter referred to as assay buffer, for 90 min at 37°C with shaking. The plates were washed six times in PBS+0.05% Tween 20 (polyoxyethylene sorbitan monolaurate, Sigma P-1379), hereafter referred to as wash solution. Antisera samples from immunized mice were serially diluted in the range of 1:100 to 1:32,000, or supernatants from hybridoma cell cultures were added to the wells and incubated for 90 minutes at 37°C with shaking, then washed six times with washing solution. 100 µl of horseradish peroxidase-APA (HRPAPA) conjugated antibody raised in goat against mouse Fab was diluted 1:1200 and added to the wells and incubated for 90 minutes at 37°C with shaking, and then washed six times with washing solution. 100 µl of substrate solution (dissolved one tablet of OPD and one tablet of H2O2 in 20 ml of water) was added to the wells and incubated at room temperature for 30 minutes. The enzyme reaction was stopped by adding 100 µl of 4 N HCl per well. Absorbance in the 96-well plates was read in an ELISA reader at 492 and 405 nm, and the results were calculated using a four-parameter logistic algorithm on a PC connected to the ELISA reader, using MultiCalc software. Example 4 Fusion, hybridoma preparation, clone selection and antibody purification from ascites fluids The fusion procedure and hybridoma cell selection were performed according to the procedure described by Eshnar, Z. (1985). Briefly, spleen cells from M-1 mice, which had been revaccinated 2-4 days before fusion, were fused with myeloma cells using a short incubation with PEG. The PEG was slowly diluted with DMEM and then completely removed by centrifugation. The cells were HU 226 194 Β1 were resuspended in DMEM-HAT medium, distributed into 96-well plates at a concentration of approximately 3.4* 10~4 cells / well, and incubated for 10-14 days in an incubator containing 8% carbon dioxide at 37°C. The medium in all wells containing hybridomas was replaced with DMEM supplemented with 10% horse serum (HS) within 10 days. Samples taken from the hybridoma culture supernatant were screened for the presence of Mabs specific for r-hsLDLR using the direct ELISA described in Example 3. DMEM containing assay buffer and 10% HS was used in the blind experiments, C7-Mab (sold by Amersham) and M-1 mouse antiserum were used as positive controls, while soluble p55-TNF receptor-specific monoclonal antibody was used as negative control.Cells from wells where antibodies were detected in the culture supernatant were transferred to 24-well plates and then to 25 cm2 T-plates. Expanded cultures were monitored for secretion of r-hsLDLR-specific Mabs. Cells from positive cultures were frozen in vials and stored in liquid nitrogen. A total of approximately 1000 cultures were screened for r-hsLDLR antibodies. The 54 cultures with the highest immunoreactivity were retested several times. The five cultures with the highest activity (12, 28, 29, 30 and 50) were cloned by limiting dilution in 96-well plates. Supernatants from the growing clones were tested several times for r-hsLDLR antibodies using direct ELISA. Cells from positive hybridoma clones were grown in tissue culture dishes containing DMEM supplemented with 15% horse serum, and a portion of the cultures was frozen in ampoules. In parallel, cells from different hybridoma clones were injected into 2-4 mice to prepare ascites fluid. Antibodies were purified from ascites fluid by ammonium sulfate precipitation or protein G column. Briefly, 7.5 ml of ascites fluid was diluted 1:3 in 20 mM phosphate buffer (pH=7) and applied to a 5 ml protein G column (C10 / 10). The column was washed with 20 mM phosphate buffer (pH=7), and the Mabs were eluted with 100 mM glycine buffer (pH=2.7). The pH in the eluted fractions was adjusted to 7-7.5 with 1 M Tris buffer (pH=9.3). Example 5 Screening of Mab Pairs for ELISA and Optimization of ELISA Parameters Mabs purified from ascites fluids as described in Example 4 were used in a matrix-based series of experiments to select the best Mab pair for use as primary and secondary antibodies in a sandwich ELISA for measuring r-hsLDLR as described in Example 6, below. Briefly, 96-well plates were coated with ascites fluids from five hybridomas (# 12, 28.28, 29.08, 30, and 50.05) purified by ammonium sulfate precipitation or protein G column. The antibodies were screened with the +291 form of r-hsLDLR produced in CHO cells as antigen, as well as its +292 form (containing amino acids from Asp+4 to Cys+292) and +331 form (containing amino acids from Asp+4 to Cys+331).1 ml of each of the partially purified Mabs listed above were labeled with biotin to screen their sensitivity as secondary antibodies in a sandwich ELISA. Briefly, 1.5 mg of ammonium sulfate-precipitated purified Mabs were adjusted to pH=8.5 with 30 μΙ of 0.5 M NaHCO3. 0.75 mg of biotinOSu (N-hydroxysuccinimidobiotin) (Biotin-OSu, Sigma cat. no. H1759, 5 mg dissolved in 200 μΙ DMSO) was added to the antibody solution and incubated for two hours at room temperature with gentle shaking, then overnight at 2-8 °C. The reaction mixture was applied to a Sephadex G-25M (Pharmacia, cat. no. 17-0851-01) PD10 column to separate the biotinylated Mabs and the unreacted excess biotin-OSu. Initial preliminary experiments showed that Mabs 29.08 and 30 gave the highest signal above background when used as secondary antibodies in ELISA. The reaction of these two clones was retested as secondary antibodies, where antibodies 12, 28, 29.08 and 50 were used to coat the plates. The results of the experiment clearly showed that Mab 28 was the most suitable for coating. The best results in terms of signal intensity and specificity were obtained by coating the microtiter plate with Mab 28 and using biotin-labeled Mab 29.08 as the secondary antibody. These Mabs gave good results with all three forms of r-hsLDLR (+291, +292 and +331). The absorbance at 492 / 405 nm was approximately 1.3 OD with all three forms. The three forms of r-shLDLR antigen were analyzed in serial dilutions over a concentration range of 0.9-1000 ng / ml. Dose-response curves were obtained using Mab 28 as the coating and biotinylated Mab 29.08 as the secondary antibody. This combination gave a linear response to r-hsLDLR over a concentration range of 1-10 ng / ml. Various parameters that can influence ELISA, such as reagent concentrations, incubation times, buffers and plate selection, were optimized by testing the following parameters: The wells of microtiter plates were coated with 5-10 pg / ml Mab28 in PBS. Buffer composition: a) PBS+Tween 20 b) Tris-Ca2++NaCI+Tween 20 Blocking solutions: a) 1% gelatin, solution: PBS, 0.05% Tween, 0.005% thimerosal; b) 1% BSA, solution: PBS, 0.05% Tween, 0.005% thimerosal; HU 226 194 B1 c) 1% FBS, solution: PBS, 0.05% Tween, 0.005% thimerosal; d) 1% milk, solution: PBS, 0.05% Tween, 0.005% thimerosal; e) I Block, Hy Pulp and Hy Yeast. Secondary antibody: blot-labeled Mab 29.08, concentrations: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:10,000, equivalent to a concentration range of 10.74-0.537 pg / ml. Extravidin concentrations: 1:500, 1:1000, 1:2000, 1:4000,1:8000,1:10,000, which is equivalent to a concentration range of 400.2 pg / ml. Based on the experiments, we finally developed the method described in Example 6 below for the sandwich ELISA test. Example 6 Developed sandwich ELISA for r-hsLDLR In the sandwich ELISA for r-hsLDLR, Mabs 28 and 29 were used. Briefly, 96-well plates were coated with 100 μΙ of Protein-G-purified Mab28 (5 pg / ml) overnight at 2-8 °C or for 3 hours at 37 °C. The plates were then washed five times with PBS+0.05% Tween 20. The plates were incubated with 200 μΙ of blocking solution (PBS+1% BSA, or gelatin+0.05% Tween 20+0.005% thimerosal) for one hour at 37 °C or overnight at 4 °C and washed five times with PBS+0.05% Tween 20. Then, 100 μΙ of sample or antigen for recording the calibration curve (CHO +291 r-hsLDLR, 0.5-32 ng / ml, diluted in blocking solution) was added to the wells and incubated for 90 minutes at 37 °C with shaking. The plates were then washed five times with PBS+0.05% Tween 20. In blocking solution, 100 μΙ of biotinylated Mab-29.08 (0.67 μg / ml) was added to the wells and incubated with shaking for one hour at 37 °C. The plates were washed five times with PBS+0.05% Tween 20. 100 μΙ of a commercially available extravidin peroxidase conjugate (ExtrAvidin TM-Peroxidase BioMakor, cat. no. 0645-1) was added to the wells at a 1:10,000 dilution and incubated with shaking for one hour at 37 °C. The plates were then washed five times with PBS+0.05% Tween 20. 125 μΙ of the substrate solution mentioned above was added to each well and incubated for approximately 10 minutes until the desired color intensity was achieved. The reaction was stopped by adding 125 μΙ of 4 N HCl. The absorbance in the 96-well plates was read with an ELISA reader at wavelengths of 492 and 405 nm, and the results were calculated on a PC connected to the ELISA reader using MultiCalc software. Example 7 Isotype of monoclonal antibodies The Ig isotype of the monoclonal antibodies was determined using a commercial isotyping kit (PharMingen International) according to the manufacturer's protocol. Clones 12.6, 28 and 30 were identified as IgG, while clone 50.30 was found to belong to the IgM class. Example 8 SDS-PAGE Western blot analysis The purified r-hsLDLR +291 form and native LDLR purified from human urine were analyzed by Western blot analysis using monoclonal antibodies specific for r-hsLDLR. Briefly, 100 ng / lane of r-hsLDLR +291 form produced in CHO cells, or native hsLDLR from urine, or crude culture of TBP-1 (as a negative control) were loaded onto a 12% SDS-polyacrylamide gel under reducing conditions (40 mM DTT). Low molecular weight markers (LMW) were loaded in one lane. This set of samples was run five times. The proteins separated on the gel were transferred to nitrocellulose membranes by electroelution. The membranes were incubated in PBS containing 10% low-fat milk, 0.1% Tween 20 for 16 hours. The membranes were cut into strips and each strip was incubated for 2 hours at room temperature with one of the five selected Mabs: 12.6, 30, 50.30, 28 or 29.08 (diluted 1:4000 in ascites fluid). The membrane strips were washed with PBS containing 0.1% Tween 20 (3*15 min) and incubated for one hour with a secondary antibody, horseradish peroxidase / alkaline phosphatase-conjugated goat anti-mouse antibody (1:10,000 dilution, BioMakor), for 2 hours at room temperature. The strips were washed with PBS containing 0.1% Tween 20 (3*15 min). Positive signals were detected by enhanced chemiluminescence (ECL, Amersham). Monoclonal antibodies 12.6 and 29.8 are able to recognize both the urinary and purified r-hsLDLR +291 form in Western blot analysis (Figure 2). Mabs 28 and 30 are able to recognize the purified r-hsLDLR +291 form. Example 9 Inhibition of the antiviral activity of r-hsLDLR by monoclonal antibodies Monoclonal antibodies specifically reactive with r-hsLDLR were tested for their ability to block the antiviral activity of r-hsLDLR (+291-form) in vitro using a cytopathic effect (CPE) inhibition assay in the VSV / WISH system. WISH cells [derived from human amnion (fetal envelope)] were cultured in MEM supplemented with 10% FBS and 4 mM glutamine at 37°C in a 5% CO2 incubator. Exponentially growing cells were plated at 40,000 cells / well in 96-well tissue culture plates 24 hours before the start of the assay. The test samples and the standard were diluted and distributed into the wells containing the cells. VSV was immediately added to the wells at a multiplicity of infection (MOI) of 0.5 pfu / cell. The plates were incubated for 16-18 hours at 37°C and then washed with ethanol. The surviving cell monolayers were visualized by Gram Crystal Violet staining. Cytopathic effect was determined relative to the standard HU 226 194 Β1 by plotting the color intensity against the concentration of the standard. To analyze the neutralizing effect of the antibodies, r-hsLDLR was preincubated for 30 min at 37 °C with increasing concentrations of ascites fluid corresponding to the Mab to be tested. These solutions were then added to WISH cell cultures in 96-well microtiter plates and vesicular stomatitis virus (VSV) was added. After incubation for 18 h, VSV-mediated cell lysis was determined by staining the remaining cells with crystal violet. Cytopathic effect was determined semiquantitatively relative to the standard by plotting the color intensity (determined in an ELISA reader) against the concentration of the standard. The effect of the Mabs was tested by increasing the concentration of r-hsLDLR. As shown in Table 1, neutralizing activity was found in two Mabs (12.6 and 50.30). In the experiment presented in Table 1, the inhibitory activity of the two Mabs was tested at a 1:40 dilution of ascites fluid. At this dilution, Mab 12.6 showed slightly higher activity than Mab 50.30. This may be a result of the properties of the Mabs and may be attributed to the difference in their concentrations in ascites fluid. The inhibitory effect of the Mabs can be overcome by increasing amounts of r-hsLDLR Mabs relative to the Mabs. At a r-hsLDLR concentration of 62.5 U / ml, none of the Mabs had any effect on r-hsLDLR activity at the antibody concentration analyzed. Table 1 Inhibition of the antiviral activity of r-hsLDLR by clones 12.6 and 50.30* VSV / Mab LDLR concentration (U / ml) 0 2.5 12.5 62.5 -VSV 1.5 1.2 1.6 1.5 +VSV (0.25) 1 7(1.7) 1.7 +VSV+50.30 clone 6 (0.4) 0.88 (1.25) 1.62 +VSV+12.6 clone 5 (0.5) 0.77 7 (0.7) 1.67 *Inhibition of antiviral activity of r-hsLDLR against VSV-mediated cell lysis of WISH cells. The effect of Mab-k inhibitor was determined at a 1:40 dilution of ascites fluid. The number of viable cells in the table is shown as OD values. The numbers in parentheses correspond to one replicate performed at 0 and 12.5 U / ml LDLR concentrations. The inhibition of r-hsLDLR antiviral activity was determined by increasing concentrations of Mabs 12.6 and 50.30. Mab 12.6 inhibited the antiviral activity of r-hsLDLR (ascites fluid) by approximately 60% at a 1:40 dilution and by approximately 35% at a 1:20 500 dilution. Clone 50.30 inhibited the antiviral activity of r-hsLDLR by approximately 45%. at a dilution of 1:40, and by approximately 15% at a dilution of 1:20,500. Based on the dose / response curves obtained for both Mabs and the observation that the extent of their inhibitory effect is reduced by excess r-hsLDLR, we hypothesize that the Mabs exert their effect through binding to r-hsLDLR. Example 10 Inhibition of HCV replication with monoclonal antibodies We tested r-hsLDLR-specific Mabs for their ability to inhibit HCV replication in primary cultures of human erythrocytes. FT167 cell culture was derived from a 57-year-old male patient who underwent lobectomy (colon cancer metastasis, right lobe) for medical reasons. Primary cultures of human hepatocytes were established by a two-step collagenase perfusion method [Maurel, P., Adv. Drug Del. Rev. 22, 105-132 (1996); Pichard, L. et al., Mol. Pharmacol. 41, 1047-1055 (1992); Ferrini, JB et al., Chem. Biol. Interactions 107, 31-45 (1997)]. Cell viability was determined before plating using the Tryptan-blue exclusion test. Four million cells were plated in 3 ml of culture medium in 60 mm plastic dishes previously coated with collagen. For longer cultures, the serum-free culture medium was Williams'E, supplemented as described [Lanford, R. et al., In Vitro Cell. Dev. Biol. 25, 174-182 (1989)]. This medium was then replaced with fresh medium every 48 hours. The cultures were maintained at 37°C in a humidified atmosphere of 5% carbon dioxide and air. Under these culture conditions, human hepatocytes maintained their differentiated phenotype for at least 35 days [Ferrini, J.B.et al., Chem. Biol. Interactions 107, 31-45 (1997)], and were susceptible to HCV infection and did not inhibit viral genome replication [Fournier, C. et al., J. Gene Virol. 79, 2367-2374 (1998)]. HCV-positive serum sample: A human serum bank was established, in which sera were obtained from patients positive for anti-HCV antibodies, tested using EIA HCV 3.0 and Chiron RIBA HCV 3.0 SIA. None of the patients were co-infected with HBV or HIV. HCV RNA was measured in each serum sample using a Roche monitor and genotyped using a line probe assay (Inno-Lipa HCV II, Innogenetics). Serum samples were stored at -80°C in small aliquots to avoid freeze-thaw cycles. Sample S42 (genotype 1b; viral load: 410,000 copies / ml) was used in these experiments. For infection and subsequent treatments, hepatocyte cultures were transferred under sterile conditions to a P3 laboratory (high-throughput isolation of human infectious microorganisms). Three days after plating, when the cells had recovered from the trauma caused by isolation, hepatocytes were infected in vitro by overnight incubation with 25 μΙ of HCV-positive serum sample. HU 226 194 Β1 (S42), in 3 ml of medium. After infection, the cells were washed three times with 3 ml of fresh medium and culture was continued under normal conditions in medium used for longer culture. Cells were treated with 3 different r-hsLDLR-specific Mabs, Mab12.6, Mab28 and Mab29.8. Thirty minutes before infection, cells were exposed to 2 or 8 pg / ml of the different Mabs. Cells were then infected as described above. Control cultures were infected under similar conditions, but no antiviral treatment was applied. In parallel experiments, the same culture was treated with 5000 U / ml IFNa under similar conditions for comparison (IFNa strongly inhibits HCV replication in cells). All treatments were performed in duplicate. On day 5 post-infection, the medium was removed and the cultures were washed 3 times with cold phosphate-buffered saline. RNA was purified from 4*106 hepatocytes using a guanidium isothiocyanate / phenol extraction procedure [Chomczynski, PN and Sacchi N., Analyt. Biochem. 162, 156-159 (1987)]. The precipitated RNA was dissolved in 50 μΙ diethylpyrocarbonate (DEPC)-treated water and quantified. One pg of cellular RNA was analyzed in a strand-specific rThRT-PCR assay. To avoid possible contamination, a strand-specific RT-PCR assay was performed in three different rooms in sequence: pre-PCR room, PCR room, and post-PCR room. RNA was dissolved in 10 µl DEPC-treated water, covered with mineral oil, and heated to 95 °C for 1 min. The temperature was lowered to 70 °C, and 10 µl pre-warmed cDNA reaction mixture was added. The temperature was then lowered to 60 °C for 2 min for annealing, and the cDNA reaction was performed for 20 min at 70 °C using rTh-DNA polymerase (Perkin-Elmer). The temperature was maintained at 70 °C while pre-warmed buffer containing EGTA as a Mn2+ chelator was added to suppress rTh-RT activity. The reaction tubes were kept at 70°C until 40 µl of pre-warmed PCR mix was added.PCR was performed on a “Gene Amp® PCR-System 9600” (Perkin-Elmer) under the following conditions: initial cycle at 94 °C for 1 min, 50 cycles at 94 °C for 15 s, 58 °C for 30 s, 72 °C for 30 s, and a final extension step at 72 °C for 7 min. The nucleotide sequence of the reverse primer P3 used for the positive-strand HCV RNA assay was: 5'-TGG / ATGCACGGTCTACGAGACCTC-3' (nucleotides 342-320), and the nucleotide sequence of the forward primer P4 was: 5'-CACTCCCCTGTGAGGAACT-3' (nucleotides 38-56) [Laskus, T. et al., J. Gén. Virol. 78, 2747-2750 (1997)]. The same primers were used in reverse order to detect the negative strand. One-tenth of the amplified product was analyzed by agarose gel electrophoresis (2%), then stained with BET and photographed under UV light.In all experimental series, synthetic HCV-RNA (+) and (-) strands were diluted and 1 pg of liver-derived total RNA was added to simulate the conditions of cultured hepatocyte analysis. These mixtures were used as positive controls in the RT-PCR assay and analysis. Figure 3 shows that HCV negative strand production was completely inhibited in FT167 culture in the presence of Mabs raised against LDLr. Therefore, viral genome replication was strongly inhibited. The results were consistent with the idea that LDL may be a receptor for HCV. Example 11 Production of LDLR-specific chimeric antibodies mRNA was purified from a hybridoma line producing LDLR-specific mAb. Specific cDNA was synthesized using oligonucleotides complementary to the 5' end of the CH1 exon from the heavy chain variable domain (oligo 1) and the 5' end of the Ck hexon from the light chain variable domain, using the purified mRNA as a template. Two cDNAs were obtained, one encoding the variable region of the heavy chain (specific for LDLR) and the other encoding the variable region of the light chain (specific for LDLR). The cDNAs were cloned and sequenced. To produce the chimeric heavy chain, the gene encoding the variable region of the cloned human Ig heavy chain was replaced (using genetic manipulation) with cloned DNA encoding the variable domain of the mouse heavy chain (specific for LDLR). In the genetic manipulation, the variable region was excised from the human Ig using specific restriction enzymes and the mouse variable region was ligated. The same procedure was performed to produce the chimeric light chain. Two mammalian expression plasmids were constructed, one containing the chimeric heavy chain gene and the other the chimeric light chain gene. Both vectors were used to co-transfect the hybridoma cell line (SP6). LDLR-specific Ig production was tested by ELISA or Western blot, using the culture of transfected cells as secondary antibody. The affinity of the chimeric antibody to its ligand was monitored using Biacore. Example 12 Creation of transgenic mice containing human immunoglobulin gene loci (xenomice) and production of human mAb specific for hLDLR Xenomice were generated as described in WO 98 / 24893 and Mendez MJ et al. (Nature Genetics 15, 146-56 (1997)). We screened human-yeast artificial chromosome (YAC) libraries for YACs that HU 226 194 They contain the variable region of the human heavy chain β1 (approximately 1000 kb) (the YAC cloning method is the method of choice, requiring inserts larger than 100 kb). YACs were characterized by Southern blot analysis and Pulse Field Electrophoresis (PEGE). YACs should contain Cp, C8-Dh and Vh regions in germline configuration. Using overlapping sequences in the YACs, the YACs are recombined in yeast using a multi-step recombination strategy. Prior to recombination, the 3' end of the YAC (with the V region) is ligated to an HPRT selectable marker. The structure of the recombined YAC is confirmed by PFGE and Southern blot analysis (presence of the human heavy chain locus from the C region to the Vh region in germline configuration). The YAC acentric arm is targeted with a vector containing the entire γ2 constant region, mouse enhancer, and neomycin resistance gene, resulting in a final heavy chain containing the entire variable region, i.e., 82 Vh genes, 6 Jh genes, and 3 different constant regions (Cp, C8, Cy) with their corresponding regulatory sequences. This YAC is called yH2. This construct is used to generate the xenomouse. A similar strategy to that used above is used to generate the kappa loci, in which: a neomycin selectable marker is ligated to the reconstructed YAC containing the complete kappa loci. This YAC is called yK2. The YAC containing yH2 is introduced into ES cells by fusing YAC-containing yeast spheroplasts with HPRT-deficient E14.Tg3B mouse ES cells. HPRT-positive cells are selected. Positive clones are expanded and analyzed by Southern blot and CHEF blot analyses. Clones containing intact yH2-YAC are selected. The introduction and selection of yK2-YAC in ES cells is performed similarly to that described for yH2-YAC. YH2-containing ES cells are microinjected into C57BL / 6J mouse blastocysts. The resulting chimeric males are evaluated for germline transmission to the offspring. yH2- or yK2-transgenic mice are crossed with Dl-mice (homozygous for the targeted-inactivated mouse-derived heavy and kappa chain foci). Each yH2; Dl-transgenic strain is crossed with a yK2; Dl-transgenic strain to generate xenomouse strains. The development of B-cell production and antibody production in xenomice is evaluated by flow cytometry and ELISA. Immunization of xenomice is carried out as described in Example 2. The procedures for producing the hybridoma and screening for positive clones are similar to those described in Examples 3 and 4. Hybridoma clones 12.6, 28, 29.8, 30 and 50.30 deposited at: “Collection Nationale de Culture de Microorganismes (CNCM), Institute Pasteur, Paris”, according to the Budapest Treaty, registration numbers: I-2390,1-2391,1-2392,1-2393 and I-2394, respectively. LIST OF REFERENCES Agnello, V., Ábel, G., Elfahal, M., Knight, G. B., and Zhang, Q. X. (1999). „Hepatitis C vírus and other flaviviridae viruses enter cells via low density lipoprotein receptor [In Process Citationj.” Proc. Natl. Acad. Sci. U S A, 96(22), 12 766-71. Beisiegel, U., Schneider, W. J., Goldstein, J. L., Anderson, R. G., and Brown, M. S. (1981). „Monoclonal antibodies to the low density lipoprotein receptor as probes fór study of receptor-mediated endocytosis and the genetics of família! hypercholesterolemia.” J. Bioi. Chem., 256(22), 11 923-31. Bieri, S., Djordjevic, J. T., Daly, N. L., Smith, R., and Kroon, P. A. (1995). „Disulfide bridges of a cysteinerich repeat of the LDL receptor ligand-binding domain.” Biochemistry, 34(40), 13 059-65. Brown, M. S., and Goldstein, J. L. (1976). „Familial hypercholesterolemia: A genetic defect in the low-density lipoprotein receptor.” N. Engl. J. Med., 294(25), 1386-90. Brown, M. S„ and Goldstein, J. L. (1986). „A receptor-mediated pathway fór cholesterol homeostasis.” Science, 232(4746), 34-47. Chomczynski, P. N., And Sacchi, N. (1987). „Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction”. Analyt. Biochem. 162:156-9. Eshhar Z, 1985 „Monoclonal Antibody Strategy and Techniques in „Hybridoma technology in the bioscience and medicine, Edited by Timothy A. Springer (Plenum Publishing Corporation, 1985; Chapter 1). Fischer, D. G., Tál, N., Novick, D., Barak, S., and Rubinstein, M. (1993). „An antiviral soluble form of the LDL receptor induced by interferon”. Science, 1262 (5131), 250-3. Fischer, D. G., Novick, D., Cohen, B, Rubinstein, M. (1994). „Isolation and characterization of a soluble form of the LDL receptor, an interferon-induced antiviral protein”. Proc. Soc. Exp. Bioi. Med. 206(3), 228-32. Ferrini, J. B., Pichard, L., Domergue, J., and Maurel, P. (1997). „Long-term primary cultures of aduit humán hepatocytes”. Chem-Biol. Interactions 107:31-45. Foumier, C., Sureau, C., Coste, J., Ducos, J., Pageaux, G., Larrey, D., Domergue, J., and Maurel, P. (1998). „In vitro infection of aduit normál humán hepatocytes in primary culture by hepatitis C vírus”. J. Gén. Virol. 79:2367-74. Goldstein, J. L., Anderson, R. G., and Brown, M. S. (1979). „Coated pits, coated vesicles, and receptor-mediated endocytosis.” Natúré, 279(5715), 679-85. Goldstein, J. L., Dana, S. E., Brunschede, G. Y., and Brown, M. S. (1975). „Genetic heterogeneity in familial hypercholesterolemia: evidence fór two different mutations affecting functions of low-density lipoprotein receptor.” Proc. Natl. Acad. Sci. USA, 72(3), 1092-6. Lanford R. E., Carey, K. D., Estlack, L. E., Smith, G. C., and Hay, R. V. (1989) „Analysis of plasma protein HU 226 194 Β1 and lipoprotein synthesis in long-term primary cultures of baboon hepatocytes maintained in serum-free médium In Vitro Cell. Dev. Bioi. 25:174-82. Laskus T., Radkowski, M., Wang, L. F., Cianciara, J., Vargas, H., and Rakela, J. (1997). „Hepatitis C vírus negative strand RNA is nőt detected in peripheral blood mononuclear cells and viral sequences are identical to those in serum: a case against extrahepatic replication”. J. Gén. Virol. 78:2747-50. Maurel P. (1996) „The use of aduit humán hepatocytes in primary culture and other in vitro systems to investigate drug metabolism in mán”. Adv. Drug Del. Rév. 22:105-132. Mendez, Μ. M„ Green, LL, Corvalan, JRF, Jia XC., Maynard-Currie, EE, Yang, MH, Davis, CG, Zsebo, KM and Jakobovits, A. (1997). Natúré Genetics, 15, 146-56. Pichard, L., Fabre, I., Daujat, M., Domergue, J., Joyeux, H., and Maurel, P. (1992). „Effect of corticosteroids on the expression of cytochromes P450 and on cyclosporin A oxidase activity in primary cultures of human hepatocytes”. Mol. Pharmacol. 41:1047-55. Riachmann, L., Clark, M., Waldmann, H., and Winter, G. (1988). "Reshaping human antibodies for therapy. Natúré, 332, 323-27. Sudhof, T. C., Goldstein, J. L., Brown, M. S., and Russell, D. W. (1985). „The LDL receptor gene: a mosaic of exons shared with different proteins. Science, 228(4701),815-22. Urlaub, G. and Chasin, L. A. (1980) Isolation of Chinese Hamster Cell Mutants Deficient in Dihydrofolate Reductase Activity. Proc. Natl. Acad. Sci. USA 77: 4216-4220.

Claims

1. A monoclonal antibody, chimeric antibody, fully humanized antibody, anti-anti-ld antibody, or fragment thereof, which is capable of specifically recognizing and binding human soluble LDL receptor and fragments thereof, is capable of inhibiting hepatitis C virus replication, and which can be produced by immunizing animals with human soluble LDLR+291, i.e., a form of the human soluble receptor comprising the amino acid sequence from Asp+4 to Glu+291 of the human soluble LDLR sequence.

2. The monoclonal antibody according to claim 1, which is a monoclonal antibody expressed by hybridoma clone 12.6 deposited at the CNCM under accession number I-2390.

3. The monoclonal antibody of claim 1, which is a monoclonal antibody expressed by hybridoma clone 28 deposited at the CNCM under accession number 1-2391.

4. The monoclonal antibody of claim 1, which is a monoclonal antibody expressed by hybridoma clone 29.8 deposited at the CNCM under accession number I-2392.

5. The monoclonal antibody of claim 1, which is a monoclonal antibody expressed by hybridoma clone 30 deposited at the CNCM under accession number I-2393.

6. The monoclonal antibody of claim 1, which is a monoclonal antibody expressed by hybridoma clone 50.30 deposited at the CNCM under accession number I-2394.

7. The monoclonal antibody according to any one of claims 1-6, which is an immunoglobulin of the IgG or IgM isotype.

8. A method for detecting and / or determining the amount of human soluble LDLR, characterized in that during the performance of the method a monoclonal antibody according to any one of claims 1-7 is used.

9. Hybridoma clone 12.6, deposited at the CNCM under accession number I-2390.

10. Hybridoma clone 28, deposited at the CNCM under accession number 1-2391.

11. Hybridoma clone 29.8, deposited at the CNCM under accession number I-2392.

12. Hybridoma clone 30, deposited at the CNCM under accession number i-2393.

13. Hybridoma clone 50.30, deposited at the CNCM under accession number I-2394.

14. A method for producing a monoclonal antibody, characterized in that a cloned hybridoma comprising a splenocyte and a homogeneous or heterogeneous lymphoid cell derived from a mammal immunized with highly purified human soluble LDLR+291 - i.e., a form of the human soluble receptor containing the amino acid sequence from Asp+4 to Glu+291 of the human soluble LDLR sequence - is grown in a liquid medium or in the peritoneal cavity of a non-human mammal, allowing the hybridoma to produce and accumulate the monoclonal antibody, and recovering the produced monoclonal antibody.

15. A method for purifying human LDLR, characterized in that a material containing crude human LDLR is contacted with a monoclonal antibody according to any one of claims 1-7 or produced by the method according to claim 14.

16. An in vitro method for inhibiting the replication of hepatitis C virus, characterized in that cells are contacted with a monoclonal antibody according to any one of claims 1-7 prior to infection with hepatitis C virus, thereby inhibiting the replication of hepatitis C virus in the cells.

17. Use of a monoclonal antibody according to any one of claims 1-7 for the manufacture of a medicament for the treatment of hepatitis C virus infection.

18. A monoclonal antibody according to any one of claims 1-7, for use as a medicament.